Evaluation of Fluoroquinolone Resistance in Clinical Avian Pathogenic Escherichia coli Isolates from Flanders (Belgium).

Fluoroquinolones are frequently used antimicrobials for the treatment of avian pathogenic Escherichia coli (APEC) infections. However, rapid development and selection of resistance to this class of antimicrobial drugs is a significant problem. The aim of this study was to investigate the occurrence and mechanisms of antimicrobial resistance against enrofloxacin (ENRO) in APEC strains in Flanders, Belgium. One hundred and twenty-five APEC strains from broilers with clinical colibacillosis were collected in Flanders from November 2017 to June 2018. The minimum inhibitory concentration (MIC) of all strains and the mutant prevention concentration (MPC) of a sample of sensitive isolates were determined using a commercial gradient strip test and via the agar dilution method, respectively. Non-wild type (NWT) isolates were further characterized using polymerase chain reaction (PCR), gel electrophoresis and gene sequencing. Forty percent of the APEC strains were NWT according to the epidemiological cut-off (ECOFF) measure (MIC > 0.125 µg/mL). With respect to clinical breakpoints, 21% were clinically intermediate (0.5 ≤ MIC ≤ 1 µg/mL) and 10% were clinically resistant (MIC ≥ 2). The MPC values of the tested strains ranged from 0.064 to 1 µg/mL, resulting in MPC/MIC ratios varying from 4 to 32. The majority (92%) of the NWT strains carried one or two mutations in gyrA. Less than a quarter (22%) manifested amino acid substitutions in the topoisomerase IV parC subunit. Only three of the NWT strains carried a mutation in parE. Plasmid mediated quinolone resistance (PMQR) associated genes were detected in 18% of the NWT strains. In contrast to the relatively large number of NWT strains, only a small percentage of APEC isolates was considered clinically resistant. The most common MPC value for sensitive strains was 0.125 µg/mL. Some isolates showed higher values, producing wide mutant selection windows (MSW). Chromosomal mutations in DNA gyrase and topoisomerase IV were confirmed as the main source of decreased antimicrobial fluoroquinolone susceptibility, de-emphasizing the role of PMQR mechanisms.

Agreement of Quantitative and Qualitative Antimicrobial Susceptibility Testing Methodologies: The Case of Enrofloxacin and Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the causal agent of colibacillosis, one of the most common bacterial infections in the poultry sector. Antimicrobial susceptibility testing (AST) is essential for rational and prudent antimicrobial therapy. Subsequently, uniformity in test results from the various testing methodologies used in diagnostic laboratories is pivotal. The aim of this study was therefore to evaluate the agreement between different AST methods in determining fluoroquinolone resistance in APEC. Twenty APEC isolates were selected and subjected to four different susceptibility tests: the quantitative microbroth dilution, agar dilution and gradient strip tests, and the qualitative disk diffusion method. The experiments were performed in triplicate. Categorical agreement, essential agreement and different errors were assessed. Moreover, agreement was also evaluated by calculating intraclass correlation coefficients (ICCs) for the quantitative tests and determining the Pearson correlation coefficients for the agreement between the disk diffusion method and the quantitative tests. Categorical agreement and essential agreement when compared with the microbroth technique ranged from 85-95% and 85-100%, respectively. No very major errors (false susceptible) and only one major error (false resistant) and minor errors (results involving an intermediary category) were detected. The calculated ICC values of the three quantitative tests fluctuated around 0.970 (range 0.940-0.988). There was a high negative correlation between the disk diffusion method and the other tests (correlation coefficients ranging from -0.979 to -0.940), indicating a clear inverse relationship between the minimum inhibitory concentration value and the zone diameter of growth inhibition. In conclusion, the overall agreement between the four different testing methodologies was very high. These results confirm the reliability of the disk diffusion and gradient strip test methods as substantiated alternatives, next to the gold standard agar and microbroth dilution, for fluoroquinolone susceptibility testing of APEC isolates.

Identification of fungal isolates by MALDI-TOF mass spectrometry in veterinary practice: validation of a web application.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable method to identify fungal isolates. The success of this approach relies on the availability of exhaustive databases, but the latter were built with a focus on human pathogens. We assessed a large in-house database of reference spectra and a dedicated web application for their suitability for use in veterinary laboratories. A panel of 290 mold and yeast isolates representing 69 different fungal species was isolated from various animals (including pets, cattle, and zoo animals) and identified using both MALDI-TOF MS and conventional techniques. The performance of the 2 methods was compared, and identifications were confirmed by DNA sequencing. MALDI-TOF MS allowed distinction between some closely related species and achieved 89% correct identification at the species level. In comparison, only 60% of the isolates were correctly identified with conventional approaches. Using this online application, MALDI-TOF MS thus appears to be a relevant alternative for the identification of fungal isolates encountered by animal health professionals.

Quantitative Outcomes of a One Health approach to Study Global Health Challenges.

Having gained momentum in the last decade, the One Health initiative promotes a holistic approach to address complex global health issues. Before recommending its adoption to stakeholders, however, it is paramount to first compile quantitative evidence of the benefit of such an approach. The aim of this scoping review was to identify and summarize primary research that describes monetary and non-monetary outcomes following adoption of a One Health approach. An extensive literature search yielded a total of 42,167 references, of which 85 were included in the final analysis. The top two biotic health issues addressed in these studies were rabies and malaria; the top abiotic health issue was air pollution. Most studies described collaborations between human and animal (n = 42), or human and environmental disciplines (n = 41); commonly reported interventions included vector control and animal vaccination. Monetary outcomes were commonly expressed as cost-benefit or cost-utility ratios; non-monetary outcomes were described using disease frequency or disease burden measurements. The majority of the studies reported positive or partially positive outcomes. This paper illustrates the variety of health challenges that can be addressed using a One Health approach, and provides tangible quantitative measures that can be used to evaluate future implementations of the One Health approach.

Low prevalence of human enteropathogenic Yersinia spp. in brown rats (Rattus norvegicus) in Flanders.

Brown rats (Rattus norvegicus) have been identified as potential carriers of Yersinia enterocolitica and Y. pseudotuberculosis, the etiological agents of yersiniosis, the third most reported bacterial zoonosis in Europe. Enteropathogenic Yersinia spp. are most often isolated from rats during yersiniosis cases in animals and humans, and from rats inhabiting farms and slaughterhouses. Information is however lacking regarding the extent to which rats act as carriers of these Yersinia spp.. In 2013, 1088 brown rats across Flanders, Belgium, were tested for the presence of Yersinia species by isolation method. Identification was performed using MALDI-TOF MS, PCR on chromosomal- and plasmid-borne virulence genes, biotyping and serotyping. Yersinia spp. were isolated from 38.4% of the rats. Of these, 53.4% were designated Y. enterocolitica, 0.7% Y. pseudotuberculosis and 49.0% other Yersinia species. Two Y. enterocolitica possessing the virF-, ail- and ystA-gene were isolated. Additionally, the ystB-gene was identified in 94.1% of the other Y. enterocolitica isolates, suggestive for biotype 1A. Three of these latter isolates simultaneously possessed the ail-virulence gene. Significantly more Y. enterocolitica were isolated during winter and spring compared to summer. Based on our findings we can conclude that brown rats are frequent carriers for various Yersinia spp., including Y. pseudotuberculosis and (human pathogenic) Y. enterocolitica which are more often isolated during winter and spring.

Impact of the sampling method and chilling on the Salmonella recovery from pig carcasses.

Differences in recovery of Salmonella on pig carcasses using non-destructive and destructive sampling methods is not well understood in respect to the chilling processes applied in slaughterhouses. Therefore, in two slaughterhouses, four strains at two different concentrations were inoculated onto pork skin. Inoculated skin samples were sampled before and after chilling with two sampling methods: swabbing and destruction. Both slaughterhouses were visited three times and all tests were performed in triplicate. All samples were analysed using the ISO-method and recovered isolates were confirmed by PFGE. The chilling system (fast or conventional cooling) nor the sampling step (before and after chilling) did not significantly influence the recovery of Salmonella. However, swabbing after chilling leads to an underestimation of the real number of contaminated carcasses. Therefore, destructive sampling is the more designated sampling method after chilling.

Association between microbiological and serological prevalence of human pathogenic Yersinia spp. in pigs and pig batches.

Pigs are the main reservoir of human pathogenic Y. enterocolitica, and the microbiological and serological prevalence of this pathogen differs between pig farms. The infection status of pig batches at moment of slaughter is unknown while it is a possibility to classify batches. A relation between the presence of human pathogenic Yersinia spp. and the presence of antibodies could help to predict the infection of the pigs prior to slaughter. Pigs from 100 different batches were sampled. Tonsils and pieces of diaphragm were collected from 7047 pigs (on average 70 pigs per batch). The tonsils were analyzed using a direct plating method and the meat juice collected from the pieces of diaphragm was analyzed by Enzyme Linked ImmunoSorbent Assay. The microbiological and serological results were compared using a mixed-effects logistic regression at pig and batch level. Yersinia spp. were found in 2031 (28.8%) pigs, antibodies were present in 4692 (66.6%) pigs. According to the logistic regression, there was no relation at pig level between the presence of Yersinia spp. in tonsils and the presence of antibodies. Contrarily, at batch level, a mean activity value of 37 Optical Density (OD)% indicated a Yersinia spp. positive farm and the microbiological prevalence in pig batches could be estimated before shipment to the slaughterhouse. This offers the opportunity to classify batches based on their potential risk to contaminate carcasses with human pathogenic Yersinia spp.

Assessment of Risk Factors for a High Within-Batch Prevalence of Yersinia enterocolitica in Pigs Based on Microbiological Analysis at Slaughter.

The purpose of the current study was to find farm-level factors influencing the bacteriological prevalence of Yersinia enterocolitica in pigs at time of slaughter. On 100 farms, data concerning a broad range of farm aspects (e.g., management and housing system, biosecurity, and hygiene measurements) were collected using a face-to-face questionnaire. At the slaughterhouse, tonsils of on average 70 slaughter pigs per batch were sampled to determine the infection status of pigs. After univariable mixed-effect logistic regressions, variables that were related to the Yersinia prevalence (p<0.05) were included in a multivariable model. In this model, the factors remaining positively associated with a higher Y. enterocolitica carriage in the tonsils (p<0.1) were an increasing number of piglet suppliers, a high density of pig farms in the area, and the use of semislatted floors in the fattening pig unit. The proper use of a disinfection bath before entering the stables and a poor biosecurity level were protective factors, although a higher prevalence was associated with a significant positive interaction between the presence of pets in the stables and a poor biosecurity level. Reducing the number of piglet suppliers, using a disinfection bath properly, and prohibiting pets inside the stables could be easily implemented by pig farmers to lower the prevalence of Y. enterocolitica in pigs at slaughter.

Seroprevalence of enteropathogenic Yersinia spp. in pig batches at slaughter.

Enteropathogenic Yersinia spp. are one of the main causes of foodborne bacterial infections in Europe. Slaughter pigs are the main reservoir and carcasses are contaminated during a sub-optimal hygienically slaughtering-process. Serology is potentially an easy option to test for the Yersinia-status of the pig (batches) before slaughter. A study of the variation in activity values (OD%) of Yersinia spp. in pigs and pig batches when applying a serological test were therefore conducted. In this study, pieces of the diaphragm of 7047 pigs, originating from 100 farms, were collected and meat juice was gathered, where after an enzyme-linked immunosorbent assay (ELISA) Pigtype Yopscreen (Labor Diagnostik Leipzig, Qiagen, Leipzig, Germany) was performed. The results were defined positive if the activity values exceeded the proposed cut-off value of 30 OD%. Results at pig level displayed a bimodal-shaped distribution with modes at 0-10% (n=879) and 50-60% (n=667). The average OD% was 51% and 66% of the animals tested positive. The within-batch seroprevalence ranged from 0 to 100% and also showed a bimodal distribution with modes at 0% (n=7) and 85-90% (n=16). On 7 farms, no single seropositive animal was present and in 22 farms, the mean OD% was below 30%. Based on the results obtained at slaughter, 66% of the pigs had contact with enteropathogenic Yersinia spp. at farm level. The latter occurred in at least 93% of the farms indicating that most farms are harboring enteropathogenic Yersinia spp.

Relation between serology of meat juice and bacteriology of tonsils and feces for the detection of enteropathogenic Yersinia spp. in pigs at slaughter.

The association between positive serology and culture detection of Yersinia spp. in individual pigs was determined. Pieces of diaphragm from 370 pig carcasses were collected for serological analysis, and tonsils and feces of the same carcass were collected for bacteriological analysis. Detection of anti-Yersinia antibodies in meat juice samples was done using an indirect enzyme-linked immunosorbent assay (ELISA) based on Yops (Yersinia outer proteins). Tonsils and feces were tested for the presence of enteropathogenic Yersinia spp. by direct plating on cefsulodin-irgasan-novobiocin agar plates. Of the 370 meat juice samples, 241 (65.1%) gave a positive serological reaction using a cutoff value of 20%. Enteropathogenic Yersinia spp. (Yersinia enterocolitica serotype O:3 and Yersinia pseudotuberculosis) were found in tonsils of 161 pigs and feces of 30 pigs. Recovery of enteropathogenic Yersinia from the tonsils was highly correlated with positive serotiters, whereas no correlation was found between serology and fecal excretion. Results demonstrated that serology has an acceptable sensitivity, but a relatively low specificity for the rapid detection of enteropathogenic Yersinia spp. in tonsils of pigs at slaughter.

Within-batch prevalence and quantification of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis in tonsils of pigs at slaughter.

Yersiniosis is a common bacterial zoonosis in Europe and healthy pigs are known to be the primary reservoir of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis. However, little information is available about the prevalence of these pathogens within pig batches at time of slaughter. The tonsils of 7047 fattening pigs, belonging to 100 farms, were aseptically collected immediately after evisceration in two Belgian slaughterhouses. The batch size varied between 70 and 930 pigs. On average, 70 pigs were sampled per batch. The tonsils were examined by direct plating on cefsulodin-irgasan-novobiocin (CIN) agar plates and the number of suspect Yersinia colonies was counted. Pathogenic Y. enterocolitica serotype O:3 were found in tonsils of 2009 pigs (28.5%), originating from 85 farms. The within-batch prevalence in positive farms ranged from 5.1 to 64.4%. The number of Y. enterocolitica in positive pigs varied between 2.01 and 5.98 log10 CFU g(-1) tonsil, with an average of 4.00 log10 CFU g(-1) tonsil. Y. pseudotuberculosis was found in seven farms, for which the within-batch prevalence varied from 2 to 10%. In five of these farms, both Y. enterocolitica and Y. pseudotuberculosis were simultaneously present. Human pathogenic Yersinia spp. are widespread in slaughter pig batches in Belgium as 87% of the tested batches were infected with these pathogens at the time of slaughter. The large variation of the prevalence between batches may lead to different levels of contamination of carcasses and risks for public health.